GSM6932670: csm212-2; Mycolicibacterium smegmatis MC2 155; RNA-Seq
Mycobacterium smegmatis cells were lysed by bead-beating in Trizol reagent (Invitrogen) and then RNA was isolated by chloroform extraction. RNA samples were treated with DNAse using the TURBO DNA-free kit (Invitrogen). Libraries were prepared with 500ng of total RNA. Ribosomal RNA was blocked using FastSelect reagents (Qiagen) during cDNA synthesis. RNA was fragmented in reverse transcriptase buffer with FastSelect reagent and heating to 94 degrees for 5 minutes, 75 degrees for 2 minutes, 70 degrees for 2 minutes, 65 degrees for 2 minutes, 60 degrees for 2 minutes, 55 degrees for 2 minutes, 37 degrees for 5 minutes, 25 degrees for 5 minutes. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer's instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 15 cycles using primers incorporating unique dual index tags.